Spontaneously evolved progenitor niches escape Yap oncogene addiction in advanced pancreatic ductal adenocarcinomas.

Lineage plasticity has been proposed as a major source of intratumoral heterogeneity and therapeutic resistance. Here, by employing an inducible genetic engineered mouse model, we illustrate that lineage plasticity enables advanced Pancreatic Ductal Adenocarcinoma (PDAC) tumors to develop spontaneous relapse following elimination of the central oncogenic driver - Yap. Transcriptomic and immunohistochemistry analysis of a large panel of PDAC tumors reveals that within high-grade tumors, small niches of PDAC cells gradually evolve to re-activate pluripotent transcription factors (PTFs), which lessen their dependency on Yap. Comprehensive Cut&Tag analysis demonstrate that although acquisition of PTF expression is coupled with the process of epithelial-to-mesenchymal transition (EMT), PTFs form a core transcriptional regulatory circuitry (CRC) with Jun to overcome Yap dependency, which is distinct from the classic TGFb-induced EMT-TF network. A chemical-genetic screen and follow-up functional studies establish Brd4 as an epigenetic gatekeeper for the PTF-Jun CRC, and strong synergy between BET and Yap inhibitors in blocking PDAC growth.

Recurrent repeat expansions in human cancer genomes.

Expansion of a single repetitive DNA sequence, termed a tandem repeat (TR), is known to cause more than 50 diseases1,2. However, repeat expansions are often not explored beyond neurological and neurodegenerative disorders. In some cancers, mutations accumulate in short tracts of TRs, a phenomenon termed microsatellite instability; however, larger repeat expansions have not been systematically analysed in cancer3-8. Here we identified TR expansions in 2,622 cancer genomes spanning 29 cancer types. In seven cancer types, we found 160 recurrent repeat expansions (rREs), most of which (155/160) were subtype specific. We found that rREs were non-uniformly distributed in the genome with enrichment near candidate cis-regulatory elements, suggesting a potential role in gene regulation. One rRE, a GAAA-repeat expansion, located near a regulatory element in the first intron of UGT2B7 was detected in 34% of renal cell carcinoma samples and was validated by long-read DNA sequencing. Moreover, in preliminary experiments, treating cells that harbour this rRE with a GAAA-targeting molecule led to a dose-dependent decrease in cell proliferation. Overall, our results suggest that rREs may be an important but unexplored source of genetic variation in human cancer, and we provide a comprehensive catalogue for further study.

Cost benefit analysis of survey methods for assessing intertidal sediment disturbance: A bait collection case study.

Coastal management requires cost-effective, yet accurate, assessments of habitat condition, especially in areas protected by statutory conservation measures. Unmanned Aerial Vehicles (UAVs) provide alternatives to manned aircraft and walk-over (WO) surveys. To support coastal managers with method selection, we compare the costs and benefits of the three techniques using the extent of bait collection (sediment scarring from manual digging) on intertidal mudflats from three UK sites. UAV and WO surveys were conducted in parallel and aerial photography was downloaded from the Channel Coastal Observatory (CCO). Digging was digitised from estimations on foot (WO) or by manually labelling imagery with confidence assigned (UAV/CCO). Method efficacy is compared with respect to spatial coverage, control over survey time/location, spatial resolution, positioning accuracy, and area of digging detected. Personnel hours and up-front costs (e.g. training/equipment), costs for personnel time standardised by shore area, personnel risk, and environmental impact are also compared. Regarding efficacy, CCO imagery had extensive shore coverage compared to UAV and WO, however, assessments are restricted to times/locations with available imagery. Each method's resolution was sufficient to detect digging. WO achieved the highest resolution (on foot), but the lowest positioning accuracy, in contrast to accurate feature delineation on aerial imagery. An additive two-way ANOVA revealed a significantly higher percent area of 'dug' sediment (all confidence levels) recorded by UAV than WO. CCO was the most cost-effective with no fieldwork/equipment costs. UAV had the highest up-front costs, but WO was more costly for personnel hours/km2 for survey time and digitisation. For all methods, digitisation was the most time-consuming aspect. Compared to WO, UAV achieved rapid shore surveys and the CCO and UAV methods minimise personnel risks. UAV and WO both cause wildlife disturbance, with trampling an additional WO impact. With each method suited to sediment disturbance assessment, selection will depend on resources and objectives and will be aided by this holistic cost-benefit analysis. Cost-effectiveness will improve with evolving regulations that facilitate UAV use and technological developments (e.g. machine learning for disturbance detection) that could significantly expedite imagery analysis and enable broadscale assessments from CCO or satellite imagery.

Master lineage transcription factors anchor trans mega transcriptional complexes at highly accessible enhancer sites to promote long-range chromatin clustering and transcription of distal target genes.

The term 'super enhancers' (SE) has been widely used to describe stretches of closely localized enhancers that are occupied collectively by large numbers of transcription factors (TFs) and co-factors, and control the transcription of highly-expressed genes. Through integrated analysis of >600 DNase-seq, ChIP-seq, GRO-seq, STARR-seq, RNA-seq, Hi-C and ChIA-PET data in five human cancer cell lines, we identified a new class of autonomous SEs (aSEs) that are excluded from classic SE calls by the widely used Rank Ordering of Super-Enhancers (ROSE) method. TF footprint analysis revealed that compared to classic SEs and regular enhancers, aSEs are tightly bound by a dense array of master lineage TFs, which serve as anchors to recruit additional TFs and co-factors in trans. In addition, aSEs are preferentially enriched for Cohesins, which likely involve in stabilizing long-distance interactions between aSEs and their distal target genes. Finally, we showed that aSEs can be reliably predicted using a single DNase-seq data or combined with Mediator and/or P300 ChIP-seq. Overall, our study demonstrates that aSEs represent a unique class of functionally important enhancer elements that distally regulate the transcription of highly expressed genes.

A Yap-Myc-Sox2-p53 Regulatory Network Dictates Metabolic Homeostasis and Differentiation in Kras-Driven Pancreatic Ductal Adenocarcinomas.

Employing inducible genetically engineered and orthotopic mouse models, we demonstrate a key role for transcriptional regulator Yap in maintenance of Kras-mutant pancreatic tumors. Integrated transcriptional and metabolomics analysis reveals that Yap transcribes Myc and cooperates with Myc to maintain global transcription of metabolic genes. Yap loss triggers acute metabolic stress, which causes tumor regression while inducing epigenetic reprogramming and Sox2 upregulation in a subset of pancreatic neoplastic cells. Sox2 restores Myc expression and metabolic homeostasis in Yap-deficient neoplastic ductal cells, which gradually re-differentiate into acinar-like cells, partially restoring pancreatic parenchyma in vivo. Both the short-term and long-term effects of Yap loss in inducing cell death and re-differentiation, respectively, are blunted in advanced, poorly differentiated p53-mutant pancreatic tumors. Collectively, these findings reveal a highly dynamic and interdependent metabolic, transcriptional, and epigenetic regulatory network governed by Yap, Myc, Sox2, and p53 that dictates pancreatic tumor metabolism, growth, survival, and differentiation.

YAP/TAZ Inhibition Induces Metabolic and Signaling Rewiring Resulting in Targetable Vulnerabilities in NF2-Deficient Tumor Cells.

Merlin/NF2 is a bona fide tumor suppressor whose mutations underlie inherited tumor syndrome neurofibromatosis type 2 (NF2), as well as various sporadic cancers including kidney cancer. Multiple Merlin/NF2 effector pathways including the Hippo-YAP/TAZ pathway have been identified. However, the molecular mechanisms underpinning the growth and survival of NF2-mutant tumors remain poorly understood. Using an inducible orthotopic kidney tumor model, we demonstrate that YAP/TAZ silencing is sufficient to induce regression of pre-established NF2-deficient tumors. Mechanistically, YAP/TAZ depletion diminishes glycolysis-dependent growth and increases mitochondrial respiration and reactive oxygen species (ROS) buildup, resulting in oxidative-stress-induced cell death when challenged by nutrient stress. Furthermore, we identify lysosome-mediated cAMP-PKA/EPAC-dependent activation of RAF-MEK-ERK signaling as a resistance mechanism to YAP/TAZ inhibition. Finally, unbiased analysis of TCGA primary kidney tumor transcriptomes confirms a positive correlation of a YAP/TAZ signature with glycolysis and inverse correlations with oxidative phosphorylation and lysosomal gene expression, supporting the clinical relevance of our findings.

The complex entanglement of Hippo-Yap/Taz signaling in tumor immunity.

The Hippo-Yap/Taz pathway, originally identified as a central developmental regulator of organ size, has been found perturbed in many types of human tumors, and linked to tumor growth, survival, evasion, metastasis, stemness, and drug resistance. Beside these tumor-cell-intrinsic functions, Hippo signaling also plays important immune-regulatory roles. In this review, we will summarize and discuss recent breakthroughs in our understanding of how various components of the Hippo-Yap/Taz pathway influence the tumor immune microenvironment, including their effects on the tumor secretome and immune infiltrates, their roles in regulating crosstalk between tumor cells and T cells, and finally their intrinsic functions in various types of innate and adaptive immune cells. While further research is needed to integrate and reconcile existing findings and to discern the overall effects of Hippo signaling on tumor immunity, it is clear that Hippo signaling functions as a key bridge connecting tumor cells with both the adaptive and innate immune systems. Thus, all future therapeutic development against the Hippo-Yap/Taz pathway should take into account their multi-faceted roles in regulating tumor immunity in addition to their growth-regulatory functions. Given that immune therapies have become the mainstay of cancer treatment, it is also important to pursue how to manipulate Hippo signaling to boost response or overcome resistance to existing immune therapies.

Rac1-Mediated DNA Damage and Inflammation Promote Nf2 Tumorigenesis but Also Limit Cell-Cycle Progression.

Merlin encoded by the Nf2 gene is a bona fide tumor suppressor that has been implicated in regulation of both the Hippo-Yap and Rac1-Pak1 pathways. Using genetically engineered murine liver models, we show that co-deletion of Rac1 with Nf2 blocks tumor initiation but paradoxically exacerbates hepatomegaly induced by Nf2 loss, which can be suppressed either by treatment with pro-oxidants or by co-deletion of Yap. Our results suggest that while Yap acts as the central driver of proliferation during Nf2 tumorigenesis, Rac1 primarily functions as an inflammation switch by inducing reactive oxygen species that, on one hand, induce nuclear factor κB signaling and expression of inflammatory cytokines, and on the other activate p53 checkpoint and senescence programs dampening the cyclin D1-pRb-E2F1 pathway. Interestingly, senescence markers are associated with benign NF2 tumors but not with malignant NF2 mutant mesotheliomas, suggesting that senescence may underlie the benign nature of most NF2 tumors.

Immediate impact of an intensive one-week laparoscopy training program on laparoscopic skills among postgraduate urologists.

INTRODUCTION: Laparoscopic techniques are difficult to master, especially for surgeons who did not receive such training during residency. To help urologists master challenging laparoscopic skills, a unique 5-day mini-residency (M-R) program was established at the University of California, Irvine. The first 101 participants in this program were evaluated on their laparoscopic skills acquisition at the end of the 5-day experience. METHODS: Two urologists are accepted per week into 1 of 4 training modules: (1) ureteroscopy/percutaneous renal access; (2) laparoscopic ablative renal surgery; (3) laparoscopic reconstructive renal surgery; and (4) robot-assisted prostatectomy. The program consists of didactic lectures, pelvic trainer and virtual reality simulator practice, animal and cadaver laboratory sessions, and observation or participation in human surgeries. Skills testing (ST) simulating open, laparoscopic, and robotic surgery is assessed in all of the M-R participants on training days 1 and 5. Tests include ring transfer, suture threading, cutting, and suturing. Performance is evaluated by an experienced observer using the Objective Structured Assessment of Technical Skill (OSATS) scoring system. Statistical methods used include the paired sample t test and analysis of variance at a confidence level of P

Comparison of healing after cystotomy and repair with fibrin glue and sutured closure in the porcine model.

PURPOSE: We compared healing after laparoscopic cystotomy using fibrin glue, sutures, or a combination to determine whether fibrin glue can obviate the need for sutures and whether there is any detriment when glue is used in the presence of sutures. MATERIALS AND METHODS: In 24 Yorkshire pigs, a 3.5 cm vertical cystotomy was created laparoscopically and repaired as follows: Group 1--no closure; group 2--fibrin glue closure; group 3--suture repair; group 4--combined fibrin glue and suture repair. All animals had a Foley catheter for 1 week. In each group, three animals were harvested at 1 week (acute) and three animals were harvested at 6 weeks (chronic). RESULTS: Acute: Group 1--all pigs had an unhealed defect that leaked when evaluated by cystography. Groups 2, 3, 4--mean leak pressures were 80, 97, and 60 cm H(2)O (P = 0.36), respectively. Mean bladder capacity was not significantly different between groups. Chronic: No leakage seen on a cystogram at 1 week; at 6 weeks, bladders were filled at > or =95 to 100 cm H(2)O without leakage. Histologically, there was more inflammation in the acute group v chronic group pigs. In the acute group pigs repaired with glue or suture + glue, there was more inflammation and less epithelial continuity than in the suture alone group. At 6 weeks, there was no difference between groups. CONCLUSION: Fibrin glue provoked an intense inflammatory response that might have delayed healing acutely, resulting in a lower burst pressure in both scenarios in which it was used (i.e., alone or in combination with sutures). However, by 6 weeks, there did not seem to be any difference between groups either clinically or histopathologically.

Short-term impact of a laparoscopic "mini-residency" experience on postgraduate urologists' practice patterns.

BACKGROUND: To assist practicing urologists incorporate laparoscopic urology into their practice, a 5-day mini-residency (M-R) program with a mentor, preceptor, and proctor experience was established at the University of California, Irvine, and we report the initial results. STUDY DESIGN: Thirty-two urologists underwent laparoscopic ablative (n=17) or laparoscopic reconstructive (n=15) training, including inanimate model skills training, animal laboratory, and operating room observation. A questionnaire was mailed 1 to 15 months (mean, 8 months) after their M-R program, and responses were reviewed. RESULTS: A 100% response rate was achieved. The mean M-R participant age was 49 years (range 31 to 70 years). The majority of the participants (72%) had laparoscopic experience during residency training and had performed between 5 and 15 laparoscopic cases before attending the M-R program. Within 8 months after M-R, 26 participants (81%) were practicing laparoscopic surgery. Participants were performing laparoscopic radical nephrectomy (p=0.008), nephroureterectomy (p<0.0005), and pyeloplasty (p=0.008) at substantially higher rates after training. At the same time, fewer of the M-R participants were performing hand-assisted laparoscopic surgery after training (p=0.008) compared with before the M-R. Ninety-two percent of the participants indicated that they would recommend this training program to a colleague. CONCLUSIONS: A 5-day intensive laparoscopic ablative and reconstructive surgery course seems to encourage postgraduate urologists, already familiar with laparoscopy, to successfully expand the scope of their procedures to include more complex laparoscopic techniques such as nephrectomy, nephroureterectomy, and pyeloplasty into their clinical practice.